Role of hemoglobin b93cys residue in nitric oxide bioactivity

描述（由申请人提供）：血红蛋白293cys残基是保守的，最近作为红细胞如何影响血管一氧化氮（NO）代谢和功能的潜在调节剂而受到广泛关注。然而，所涉及的确切功能和机制尚不清楚。了解这种残基如何控制血管一氧化氮功能是至关重要的，因为这些机制的功能障碍可能导致许多血管病理状态。在这个提议中，我们建立了从新的小鼠模型中产生的初步数据，这些小鼠模型只表达野生型人血红蛋白或在其红细胞中293cys残基被Ala取代的人血红蛋白。具体来说，我们提供的数据表明，在生理条件下，红细胞和血红蛋白的脱氧激活亚硝酸盐还原酶活性，导致阴离子亚硝酸盐单电子还原为NO。这一过程通过控制血红蛋白氧亲和力进行变构调节，并由293cys残基有趣地调节。在这种情况下，我们提出293cys残基在将血红蛋白氧传感与亚硝酸盐衍生的no生物活性耦合在一起是至关重要的。相反，在急性炎症性疾病脓毒症中，我们提出293cys是亚硝化应激形成s -亚硝基血红蛋白（SNOHb）的靶标，这反过来有助于与该疾病相关的血管和肺功能障碍。数据显示含sno的红细胞可以刺激中性粒细胞粘附肺内皮细胞，并以不依赖于变张力调节的方式引起血管舒张。肺部炎症和肺损伤都是脓毒症的特征。本提案将通过以下假设来研究这些新概念：在急性炎症期间，293cys残基作为血管NO信号的调节剂的作用从亚硝酸盐还原酶依赖机制转变为SNOHb依赖机制，该机制将通过以下具体目标进行测试1)确定293cys调节红细胞依赖NO血管细胞信号的机制。2)确定293cys残基在体内控制亚硝酸盐还原和NO依赖性血管细胞信号传导中的作用；3)确定293cys残基在脓毒症诱导的低血压和肺部炎症中影响红细胞作用的作用。这些目标的实现将有助于深入了解红细胞如何调节no代谢的机制和新的治疗靶点。

DESCRIPTION (provided by applicant): The hemoglobin 293cys residue is conserved and received much attention recently as a potential modulator of how red cells affect vascular nitric oxide (NO) metabolism and function. However, the precise function and mechanisms involved remain unclear. Understanding how this residue controls vascular nitric oxide function is critical since dysfunction in these mechanisms may contribute to a number of vascular pathological states. In this proposal we build upon preliminary data generated from novel mouse models that express exclusively either wild-type human hemoglobin or human hemoglobin in which the 293cys residue has been replaced with an Ala in their red cells. Specifically, we present data indicating that under physiological conditions, deoxygenation of red cells and hemoglobin activates a nitrite reductase activity that results in the one-electron reduction of the anion nitrite to NO. This process is regulated allosterically by controlling hemoglobin oxygen affinity and interestingly modulated by the 293cys residue. In this context we propose that the 293cys residue is critical in coupling hemoglobin oxygen sensing with nitrite derived NO-bioactivity. In contrast, during the acute inflammatory disease Sepsis, we propose that the 293cys is a target for nitrosative stress forming S-nitrosohemoglobin (SNOHb), which in turn contributes to the vascular and pulmonary dysfunction associated with this disease. The latter is indicated by data showing SNO-containing red cells can stimulate neutrophil adhesion to pulmonary endothelial cells and elicit vasodilation in a manner that is independent on allosteric regulation. Both pulmonary inflammation and lung injury are features of sepsis. These novel concepts will be investigated in this proposal by pursuit of the hypothesis that during acute inflammation, the role of the 293cys residue as a modulator of vascular NO-signaling changes from a nitrite-reductase dependent to SNOHb dependent mechanism which will be tested via the following specific aims 1) Determine the mechanism by which 293cys regulates RBC dependent NO vascular cell signaling., 2) Determine the role of 293cys residue in controlling nitrite reduction and NO- dependent vascular cell signaling in vivo, 3) Determine the role of the 293cys residue in affecting RBC effects during Sepsis induced hypotension and pulmonary inflammation. Accomplishment of these aims will yield insights into the mechanisms novel therapeutic targets focusing on how RBCs modulate NO-metabolism. PUBLIC HEALTH RELEVANCE: Red blood cells play important roles in controlling vascular homeostasis mechanisms. We propose herein that a specific amino acid residue of the hemoglobin protein (the 293cys residue) is critical in this regard by modulating how red cells control nitric oxide function. In this proposal we aim to elucidate the specific mechanisms by which the 293cys residue controls nitric oxide function both during normal physiological conditions and during inflammation associated with the disease Sepsis and in doing so, hope to identify novel therapeutic targets and strategies.